Figure 2

Design of lentiviral vectors for NR2B manipulation in LMAN. (a) Diagram of the eGFPhNR2B vector to overexpress human NR2B. (b) Diagram of the eGFP vector for control injections. (c) Western blots showing no human NR2B protein in HEK293FT cells without transfection (Lane 1), and greater NR2B protein (* ~180 kD band) in cells transfected with the eGFPhNR2B plasmid (possible degraded or unfinished NR2B protein product is detected as an additional lower band) (Lane 2). (d) HEK293FT cells transfected with the eGFP plasmid show GFP-expressing cells labeled using a GFP antibody (green). (e) Cells transfected with the eGFP plasmid stained with an anti-NR2B antibody show no labeled cells. (f) Merged image of (d) and (e). (g) Cells transfected with the eGFPhNR2B plasmid show GFP-expressing cells (localized in cell membranes) labeled using a GFP antibody (green). (h) Cells transfected with the eGFPhNR2B plasmid show NR2B-expressing cells (localized in cell membranes) stained with anti-NR2B antibody (red). (i) Merged image of (g) and (h) showing complete overlap of the human NR2B tagged with eGFP (yellow). (j) Darkfield view of a brain section showing the injection site in LMAN. (k) Cells expressing GFP-tagged (green) human NR2B ~3 weeks after injection of the eGFPhNR2B lentivirus in LMAN. (l) Darkfield photograph of coronal sections showing drawings of LMAN and part of nidopallium that was microdissected for protein analysis. (m) Western blots showing NR2B protein and actin protein from microdissected tissues of nidopallium and LMAN in juveniles and adult animals (3 animals shown as examples). (n) Western blot comparing endogenous NR2B protein and actin in LMAN of an adult bird (Lane 1) with additional NR2B overexpression in birds with eGFPhNR2B lentivirus in LMAN ~35–40 days after surgery (Lanes 3–7; * ~180 kD NR2B protein). Protein Standard is included in Lane 2. (o) Quantification of NR2B protein levels normalized to actin levels in Western blots. Even though the values were higher in all 4 animals, protein levels were not significantly higher in nidopallium relative to LMAN in juveniles (p = 0.125) possibly due to low sample size, but were higher in nidopallium compared to LMAN in adults (p = 0.05). Band density was quantified using ImageJ and results analyzed using two sample Wilcoxon tests for comparisons among groups, or paired Wilcoxon signed-rank tests for within-group comparisons. Scale bars, 10 μm (d–i).